1. Field of the Invention
The present invention relates generally to the fields of molecular immunology and protein chemistry. More specifically, the present invention relates to a novel mucosal immunogens for use in novel vaccines.
2. Description of the Related Art
An oral immunization strategy is when the desired mucosal immunogen is genetically fused to the A2 subunit of cholera toxin (CT) that mediates association with the B subunit of CT, a potent immunoenhancing agent. An antigen selected for evaluating the oral immunogenicity of such non-toxic CTA2/B-based constructs is the saliva-binding region (SBR) of the AgI/II adhesin from the oral bacterium Streptococcus mutans. The SBR genetically linked to CTA2/B, designated SBR-CT.sup..DELTA.A1, was found to be immunogenic by the oral route and elicited high levels of secretory immunoglobulin A (S-IgA) and serum IgG antibodies to AgI/II.
Despite its great importance for mucosal defense, the S-IgA antibody response is often of relatively short duration, lasting from a few weeks in experimental animals to a few months in humans. Moreover, whether the secretory immune system is capable of anamnestic immune responses has been debated, but recent studies in mice and humans have addressed the concept of immunological memory at the mucosal surfaces. Immunological memory can be manifested as a long-lasting immune response or as a faster and more vigorous anamnestic response to re-encounter with an antigen. A desirable vaccine characteristic is the induction of prolonged immune responses, especially when the pathogenic organism is frequently encountered at mucosal surfaces, in which case a continuing level of immunity may be necessary.
IgA antibodies in external secretions protect mucosal surfaces, e.g., of the gastrointestinal and respiratory tracts, by blocking microbial adherence and colonization. Oral administration of vaccines can result in the induction of secretory immune responses after uptake of the antigen by the gut-associated lymphoid tissues, a major IgA inductive site. However, most soluble proteins are not only poor immunogens when given orally but they may induce a state of systemic unresponsiveness known as "oral tolerance". The experimental use of cholera toxin from Vibrio cholerae or the related heat-labile enterotoxin from Escherichia coli as mucosal adjuvants inhibit induction of oral tolerance and potentiates the immune responses to co-administered protein antigens.
Another strategy to overcome problems associated with oral immunization (e.g., denaturation of the protein immunogens by gastric acid and digestive enzymes, limited absorption by the intestinal mucosa, and clearance by peristalsis) as well as the need to purify a vaccine protein, involves the use of avirulent derivatives of Salmonella typhimurium as a vaccine delivery system with tropism for the gut-associated lymphoid tissues. Oral immunization with avirulent S. typhimurium expressing heterologous antigens is generally not associated with suppression but rather with stimulation of protective secretory and serum antibody responses as well as cell-mediated immune responses.
Initial adherence of Streptococcus mutans to tooth surfaces appears to be mediated largely by the 167 kDa surface fibrillar adhesin known as AgI/II (synonyms: antigen B, P1, SpaP, PAc). The adhesion domain that interacts with salivary pellicle has been located to the alanine-rich (A) repeat region in the N-terminal part of the molecule extending from the cell surface probably in an .alpha.-helical conformation. Studies on AgI/II indicated that rhesus monkeys immunized with S. mutans and protection against dental caries mounted antibody responses especially against the complete molecule rather than against AgII, which corresponds to the C-terminal one-third. These results were supported by the finding that immunization with either complete AgI/II, or the isolated AgI component (corresponding to the N-terminal two-thirds), afforded protection against caries. Thus, one approach to immunization against S. mutans-induced dental caries can be based upon the generation of an appropriate antibody response in the saliva that would inhibit the adherence of S. mutans to tooth surfaces. Human secretory IgA (S-IgA) antibodies to AgI/II inhibit such adherence. However, S-IgA antibodies in saliva and other secretions are not effectively induced by conventional parenteral immunization.
S-IgA antibodies are most effectively induced by stimulating the common mucosal immune system, for example, by enteric immunization which stimulates the gut-associated lymphoid tissues including the Peyer's patches (PP) of the small intestine. Considerable attention has been given to the development of improved procedures for the oral delivery of vaccines, one of which is coupling antigens to the nontoxic binding B subunit of cholera toxin (CT), a safe and highly immunogenic protein in humans. CTB, because of its avid binding to G.sub.M1 ganglioside, present on all nucleated cell surfaces, is readily taken up by the M cells covering PP, and passed to the underlying immunocompetent cells which initiate the mucosal IgA antibody response. Antigen-stimulated IgA-committed B cells, and corresponding T helper cells, then emigrate via draining lymphatics to the mesenteric lymph nodes (MLN) and thence via the thoracic duct to the circulation before relocating in the effector sites of mucosal immunity, such as the salivary glands. Terminal differentiation of B cells into IgA-secreting plasma cells occurs here and their product, polymeric IgA is transported through the glandular epithelium to form S-IgA. Other antigens can be coupled to CTB to generate strong mucosal IgA antibody responses to the desired antigen and that intact CT, though toxic, serves as an adjuvant that enhances the response to co-administered antigens.
The expression of foreign genes encoding immunogens of interest in avirulent derivatives of Salmonella typhimurium is used as a strategy to induce mucosal immune responses to protein Ags which are usually poor oral immunogens when administered alone. Indeed, S. typhimurium appears to be an effective antigen delivery system because of its ability to colonize the gut-associated lymphoid tissue where secretory IgA responses are initiated (1). Electron microscopy studies have shown that S. typhimurium preferentially interacts with the specialized antigen-sampling M cells overlying the Peyer's patches in the GALT (2). At these sites, antigenic stimulation of specific IgA-committed B cells results in their migration to mucosal tissues where they differentiate into IgA-secreting plasma cells, with subsequent release of secretory IgA antibodies in external secretions (3). These antibodies play an important role in the defense of mucosal surfaces, e.g., of the gastrointestinal and respiratory tracts, by inhibiting microbial adherence and colonization or invasion (4). Depending on the species and host, Salmonella organisms may disseminate to the spleen, the liver, and regional lymph nodes, take residence in macrophages, and thereby induce serum antibody and cellular immune responses (1).
The issue of whether CTB alone has mucosal adjuvant properties has been questioned especially for oral immunization (14, 19), although CTB confers a targeting property to Ags coupled to it because of its affinity for G.sub.M1 ganglioside receptors (20). If CTB possesses immunoenhancing properties, other than its carrier/targeting effect, it could also be useful as a Salmonella-expressed adjuvant, especially for proteins that are poor immunogens even when delivered by S. typhimurium. A commercially obtained CTB preparation, lacking detectable cAMP-elevating capacity, was found to potentiate in vitro antibody production against an unrelated protein antigen by stimulating the antigen-presenting function of splenic adherent cells through enhanced IL-1 production (21). An enhancing effect on antigen presentation by macrophages was also demonstrated for recombinant (r)CTB (22), which, moreover, up-regulates expression of MHC class II molecules on B cells, which can also act as antigen-presenting cells (23). The fact that commercially available CTB is contaminated with small but variable amounts of intact CT may explain conflicting reports on the adjuvant capacity of CTB (14) as well as findings that commercial CTB is superior to rCTB as an adjuvant for intranasal (i.n.) immunization (24, 25).
The prior art is deficient in the lack of effective mucosal immunogens, for use in, e.g., a caries vaccine. The present invention fulfills this longstanding need and desire in the art.